Synthesis of biocytin



United States Patent cc 2,720,527

Patented Oct. 11, 1955 Condensation of lysine "copper complex and biotin acid halide is carried out in the presence of an alkaline con 2,720,527 densing agent, such as aqueous alkali or alkaline earth metal hydroxide solutions, pyridine, and the like. It is SYNTHESIS OF BIOCYTIN 5 preferred to introduce the biotin acid halide in chloroform Donald E. Wolf, Princeton, and. Karl Folkers' and Robert solution. However, water immiscible solvents other than L. Peck, Plainfield, N. L, assignors to Merck & Co., chloroform may be employed for this purpose, provided Railway, a 'l fi New Jersey they contain no hydroxyl or amino groups which would i, i I i react withthe acid halide. Such solvents as ether and No g fi gggfl g 1951 benzene would be suitable. The copper complex of lysine is soluble in the aqueous phase of the reaction mixture. 4 Claims- The condensation is carried out at a pH of about 8 by the addition of suflicient base.

The condensation reaction results in the formation of This invention relates to a novel process for the prepara- 15 e N -(D-biotinyl)'-L-lysine copper complex which is betion of e-N-(D-biotinyll-L lysine,also known as biocytin. lieve'd to have the following. formula:

3 0 N NE HN/ NH s NE; 00 H s Biocytin is a growth-promoting substance related to The e-N-(D-biotinyl.)-L-lysine *c'opper complex is debiotin. Heretofore, it has been isolated from such sources composed by treating said compound with acid and reas autolyzed yeast extracts However, this procedure is moving the copper as' an insoluble salt. we prefer to inlong and tedious and furnishes only minute quantities troduce hydrogen sulfide into the acidified mixture to of biocytin relative to the amount of starting material. effect the latter step; Removal of the" precipitated copper It is an object of our invention to provide a process for sulfide leaves an aqueous solution of e-N-(D'-biotinyl)"-L- the preparation of e-N-(D-biotinyl)-L-lysine from readily lysine having the structural formula available starting materials. O

Regarded in certain of its broader aspects the process in accordance with our invention. involves condensing L- lysine copper complex with biotin acid halide in. the NE NH irfsence of a condenslingagegtfto forigr erN-(p-biot inynp K E E ysine copper comp ex, am 1 ying t e reaction mixture thus obtained, removing the copper as an insoluble solt, 40 E /CH (CH2)C0NH(0H) CHOOOH and recovering e-N-(D-biotinyl)-L-lysinc. S

In preparing e-N-(D-biotinyl)-L-lysine (biocytin) we utilize as one of the Starting materials L lysine copper ThlS latter product may be recovered by conventional complex. Although the structure of the complex is not q l h th definitely established at the present time, it is believed to D It lsbof course 16 to y our synt esls be represented by the following formula: or P form lysme' Lflysme was P m t 6 present instance since we wished to obtain the natural 0 form of biocytin as the end product of the reaction. (JO\ Nm\ The following example illustrates specific embodiments EN CH CH H G of our invention:

2 2)4 NH 1, u\o 0/ C( H2)4NH: EXAMPLE 2 Preparation of e-N-(D-biotinyl)-L-lysine To a boiling aqueous solution of 1.64 g. of L-lysine The above com ound ma be re ared b reactln a mono mineml acidpsalt of g f as Ldiglsine i monohydrochloride was added an excess of solid copper carbonate. The blue solution was cooled in ice and filhydrochloride, with a basic solution of a copper salt.

Copper sulfate or copper oxide may be employed in this iered' This: preiparatlo-n of the L'lyslfle copper complex is reported 1n Bi0chcm1cal Journal 37. 515 (1943). The

reaction, however, we prefer to use copper carbonate. filtrate was cooled to about C i an ice salt bath The preparation of L-lysine copper complex is reported n and a chloroform suspension of biotin acid chloride, pregi i zgf and Sanger m the Blochemlcal Journal pared from 1.0 g. of D-biotin, was added in portions over a fifteen minute period. The pH was adjusted to above pH 8 by the addition of sufiicient 2.5N sodium hydroxide solution. The mixture was stirred vigorously throughout the addition of the D-biotin acid chloride. Stirring was We have found that the L-lysine copper complex described above may be condensed with biotin acid halide having the structural formula f continued for an additional 30 minutes, while the mixture C was allowed to warm to room temperature. The mixture was then centrifuged, the blue aqueous layer removed, I 5 and the chloroform washed with water and dilute hydro- 7 chloric acid. All the washings were then combined with E2 CH-(C 2)rGOCI the original blue aqueous mother liquor, which was ad- S justed to pH 2 with hydrochloric acid.

Gaseous hydrogen sulfide is bubbled into the reaction mixture whereupon copper sulfide precipitates. The mixture is filtered through Supercel and the filtrate concentrated to dryness at reduced pressure. The residue was extracted with water and this extract was then subjected to a plate countercurrent distribution, using an organic layer of equal parts chloroform and ortho-cresol and an aqueous layer of equal volume adjusted to pH 3 with hydrochloric acid. Plates 4-9 contained most of the ninhydrin-reacting material. The contents of these plates were combined, ten volumes of petroleum ether added, and the solution extracted with water. The combined aqueous extracts were washed with ether and lyophilized. The colorless residue was dissolved in water, and acetone added to cloud the solution. e-N-(D-biotinyl)'-L-lysine was obtained as a colorless crystalline precipitate.

A quantity of 1.58 g. of crude e-N-(D-biOtlIlYD-L-IYSlHG obtained from several preparations in the above manner was subjected to a 12 plate countercurrent distribution, using the same solvent system as above. Plates 4-8 contained 715 mg. of colorless crystalline e-N-(D-biotinyD-L- lysine which on rapid recrystallization from water gave 498 mg. of crystalline product, having a melting point of 228-229 C. We have found that crystalline biocytin having a melting point of 228-229 C. was obtained when the compound was allowed to crystallize rapidly from water, aqueous acetone or aqueous methanol. However, when the crystalline biocytin was allowed to separate slowly, it was found to sinter slightly at 227 C., and melt at 245-252 with decomposition.

Analysis.-Calculated for C1sH2sN4O4S: C, 51.59; H, 7.58; N, 15.04. Found: C, 51.44; H, 7.35; N, 14.83.

It should be understood that various changes may be 1 alkaline aqueous solution of L-lysine copper complex with a suspension of biotin acid halide in a water-immiscible inert organic solvent, maintaining the pH of the reaction mixture at approximately pH 8, and the temperature at between l5 C. and room temperature to form e-N-(D- biotinyl)-L-lysine copper complex, acidifying the latter compound with an inorganic mineral acid, removing the copper as an insoluble salt and recovering e-N-(D- biotinyl L-lysine.

2. The process that comprises intimately contacting an alkaline aqueous solution of L-lysine copper complex with a suspension of biotin acid chloride in chloroform, maintaining the pH of the reaction mixture at pH 8 by the addition of sodium hydroxide at a temperature between -15 C. and room temperature to form e-N-(D-biotinyD-L- lysine copper complex, reacting the latter compound with hydrochloric acid, removing the copper as the insoluble salt and recovering e-N-(D-biotinyl)-L-lysine.

3. The process that comprises intimately contacting an alkaline aqueous solution of L-lysine copper complex with a suspension of biotin acid halide in a water-immiscible inert organic solvent at a pH of about 8 and at a temperature between l5 C. and room temperature to form s-N- (D-biotinyl) L-lysine copper complex.

4. The process that comprises intimately contacting an alkaline aqueous solution of L-lysine copper complex with biotin acid chloride suspended in chloroform, maintaining the pH of the reaction mixture at pH 8 by the addition of aqueous sodium hydroxide at a temperature between 15 C. and room temperature to form e-N-(D- biotinyl)-L-lysine copper complex.

References Cited in the file of this patent UNITED STATES PATENTS 2,462,124 Neuberg et al Feb. 22, 1949 2,519,530 Wolf Aug. 22, 1950 OTHER REFERENCES Kurtz, J.: Biol. Chem., vol. 180, pp. 1253-67 (1949).

Science, vol. 114, December 14, 1951, pp. 635-36. Wright et al.: JACS 72, February 1950, p. 1048. 

1. THE PROCESS THAT COMPRISES INTIMATELY CONTACTING AN ALKALINE AQUEOUS SOLUTION OF L-LYSINE COPPER COMPLEX WITH A SUSPENSION OF BIOTIN ACID HALIDE IN A WATER-IMMISCIBLE INERT ORGANIC SOLVENT, MAINTAINING PH OF THE REACTION MIXTURE AT APPROXIMATELY PH 8, AND THE TEMPERATURE AT BETWEEN -15* C. AND ROOM TEMPERATURE TO FORM E-N-(DBIOTINYL)-L-LYSINE COPPER COMPLEX, ACIDIFYING THE LATTER COMPOUND WITH AN INORGANIC MINERAL ACID, REMOVING THE COPPER AS AN INSOLUBLE SALT AND RECOVERING E-N-(DBIOTINYL)-L-LYSINE. 